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  1. 1.1.2 Drug Ion Electrical Properties and Drug Loading Capacity The interaction between the drug and the phospholipid layer molecule has an important effect on the structure and load of the liposome, and the effect of the charge effect is particularly significant. Generally, when the charge properties of the drug and the phospholipid molecular layer are the same, it is not easy to be encapsulated. By adding appropriate excipients during the preparation of the liposome to make it a charged liposome opposite to the charge of the encapsulated drug, the drug encapsulation rate can be improved. For example, in the preparation process, octadecylamine is added to obtain positively charged liposomes, and phosphatidic acid is added to obtain negatively charged liposomes. The antiviral drug cidofovir is negatively charged under normal physiological conditions. It is found that liposomes made with positively charged phospholipids composed of DOTAP and DC have a significantly higher encapsulation rate than liposomes made with electrically neutral phospholipids. However, other studies have shown that when the positively charged drug sumatriptan uses a neutral phospholipid as the membrane material, the encapsulation rate is low, and when the positively charged material stearylamide is added to the membrane material, the phospholipid membrane is significantly strengthened, thereby t lop09he leakage of the drug is prevented, and the drug load is increased. However, the encapsulation efficiency was lower than that of positively charged membranes after the addition of negatively charged membrane dicetyl phosphate, indicating that in some drug encapsulation processes, charge is not the main factor influencing. 1.1.3 Decoration and Encapsulation Rate of Medicinal Chemical Structure The chemical structure of a drug determines its physical and chemical properties. By modifying the structure of a drug to some extent, the hydrophilic and hydrophobic properties of the drug can be improved, thereby improving the encapsulation efficiency of the drug. Researchers reacted the anticancer drug cyclocytidine with palmitic acid to obtain two derivatives of monopalmitate and dipalmitate, which were separately encapsulated to prepare liposomes. As a result, the encapsulation rates of the two derivatives were found. It rose to 86.5% and 93.7% respectively, while the original drug was only 21%. The HLB value shows that on the one hand, the original drug is converted from hydrophilic to lipophilic after esterification, and the encapsulation position of the drug is correspondingly transferred from the aqueous phase to the external lipid phase. On the other hand, the long ester chain obtained by structural modification can be embedded in the lipid membrane. In addition, the fluidity of the lipid membrane is reduced, thereby increasing the liposome stability and encapsulation efficiency. Liposomal particle size design Particle size is an important evaluation index of liposomes, and its size and degree of uniform dispersion directly affect the in vivo behavior of liposomes. Large particle size liposomes are easily endocytosed by macrophages and concentrated in the liver. Smaller particle size liposomes can effectively prolong the circulation time of the drug and play a long-lasting effect. When the particle size is less than 50nm, liposomes can penetrate the liver endothelium and enter the spleen, bone marrow and tumor tissues. Duan Yisong et al etc. used long-circulating material polyethylene glycol to prepare mitoxantrone long-circulating liposomes with an average particle size of 60nm. Compared with ordinary liposomes, the average residence time in rabbits was prolonged by 6.2h, reflecting Its long cycle advantage. Awasthi et al. Investigated the particle size on the circulation time of PEG-modified liposomes in rabbits and found that the optimal particle size is 160-220 nm. Large particle size liposomes (400-530nm) are highly targeted to liver and spleen-enriched reticular macrophages. When the liposome particle size increases to 1-12 μm, it is easily taken up by the lungs. After azithromycin was prepared into cationic liposomes, the mice were administered tail vein to study the distribution of azithromycin in mouse whole blood and various tissues. AUC increased by 7.4 times. This is because liposomes larger than 6 μm will be mechanically filtered by pulmonary capillaries and then taken up by monocytes into lung tissue. Preparation method selection The preparation method of liposomes greatly affects the structure and particle size of liposomes, so it is generally selected according to the nature of the drug and the purpose of the drug. For fat-soluble drugs, mechanical dispersion methods such as film dispersion method and freeze-drying method can be selected to prepare multilayer liposomes with large particle diameters, so that the drug is slowly released in the target tissue. If you need to increase the drug's transport speed, you can choose to prepare small single-compartment liposomes. The main methods include ethanol injection, surfactant treatment, film-ultrasonic method, and so on. The drug loading of water-soluble components is generally not high. The key is to increase the volume of the aqueous phase in the liposome. Therefore, large monolayer or polycystic liposomes are generally selected for preparation. The reverse thin film dispersion method and the double emulsion method and the freeze-thaw method are suitable for the preparation of large particle size aqueous drug-loaded liposomes. Among them, the reverse thin film dispersion method is mostly large monolayer liposomes, including The sealing rate can reach 60-65%, and some studies have shown that for the hydrophilic drug salvianolic acid B, the large monolayer liposomes (LUV) prepared by the reverse evaporation method, the ethanol injection method, and the double emulsion method are used. Compared with liposomes, the encapsulation rate is the highest. The repeated freeze-thaw process in the freeze-thaw method will be accompanied by the formation of ice crystals, which will cause mechanical damage to the phospholipid bilayer, thereby increasing the chance of water-soluble drugs entering the phospholipid bilayer and increasing the encapsulation rate. At the same time, multi-compartment liposomes can be prepared into small single-compartment liposomes by repeated extrusion and freeze-thaw methods, which increases the drug loading space and drug loading. In addition, the microencapsulation method is suitable for preparation Small-particle-size drug-loaded liposomes in aqueous phase. For amphiphilic drugs such as weak base and weak acid, they can be encapsulated by active drug loading methods, such as PH gradient method, ammonium sulfate gradient method metal ion gradient method, and so on. In some cases, a single method alone cannot meet the requirements of encapsulation efficiency and particle size, especially for compound drugs with different properties in the co-loading concentration, so it is often combined with several methods.
  2. Mesenchymal stem cells have the characteristics of low immunogenicity and homing to ischemic or injured tissues. After entering into the host body, they can homing to specific sites and be differentiated into endoderm, mesoderm, and ectoderm under the influence of microenvironment cells derived from individual germ layers, such as bone, cartilage, tendon, fat, liver, kidney, skin, muscle, nerve, and even pancreas, are more than 10 kinds of mature cells, thus becoming ideal seed cells for organ repair in regenerative medicine. Initially, mesenchymal stem cells were found in the bone marrow, but highly invasive bone marrow donation experiments were needed. In addition, the number and differentiation potential of mesenchymal stem cells decreased with age. Recently, umbilical cord blood has been less damaged due to acquisition methods and has been used as an alternative source of mesenchymal stem cells. Another promising source of mesenchymal stem cells is adipose tissue. This review compares these three mesenchymal stem cell sources from aspects such as morphology, success rate of isolation of mesenchymal stem cells, frequency of clonal colony formation, expansion potential, multi-directional differentiation ability, and immune phenotype. Adipose tissue can be used as an alternative source of bone marrow tissue for the isolation of mesenchymal stem cells. In addition, people have also found that mesenchymal stem cells are also found in cord blood, periodontal ligaments, amniotic fluid, dermis, periosteum, skeletal muscle, fetal lung, fetal liver, placenta and pancreas. Mesenchymal stem cells have broad clinical application prospects and can be used to treat diseases of the nervous system, liver and kidney injury, autoimmune disease, heart disease, bone disease, cartilage disease, ischemic vascular disease, diabetic complications and tumors. They can also be used in tissue engineering and facial shaping. In addition, they can be co-transplanted with hematopoietic stem cells to treat blood diseases. Based on this, the article made an inventory of the research progress made by mesenchymal stem cells in recent years. 1.TEPCM: magnetic mesenchymal stem cells promise to improve cartilage repair doi: 10.1089 / ten.tec.2019.0001 Cells carrying superparamagnetic iron oxide nanoparticles (SPIOs) can be directed to a specific location by an external magnetic field, which is beneficial for tissue repair. Recently, a research report entitled "In Vitro Safety and Quality of Magnetically Labeled Human Mesenchymal Stem Cells Preparation for Cartilage Repair" was published in an international magazine Tissue Engineering Part C: Methods. The safety and effectiveness of this magnetically labeled mesenchymal stem cells (MSCs) in repairing cartilage defects. Researcher Dr. Naosuke Kamei said that “in this study, we demonstrated the safety of magnetically labeled MSCs through karyotyping, clone formation experiments, and total proliferation experiments. After labeling, we found only small differences in mesenchymal stem cells”. Researchers can evaluate the quality of stem cells by the differentiation of chondrocytes and their reactivity to magnetic forces. The results show that the appropriate concentration of superparamagnetic iron oxide nanoparticles can help optimize the mesenchyme while ensuring magnetic attractiveness and differentiation ability of plastid stem cells. Nat Commun: Identify key proteins that regulate angiogenesis in tumors doi: 10.1038 / s41467-019-10946-y Recently, in a research report published in the international journal Nature Communications, scientists from the Barcelona Institute of Biomedicine found that inhibiting the function of p38 protein or inhibiting angiogenesis in human and mouse colon cancer. This process is called angiogenesis, which is essential for cancer cell growth and can promote cancer progression and metastasis. Researcher Dr. Angel R. Nebreda said that we found that p38 activity is very important for mesenchymal stem cells (MSCs). These stem cells have high plasticity and can be concentrated around blood vessels. It participates and plays a role in many key processes, such as tumor formation. This study clarifies the molecular mechanism of tumor angiogenesis. Researchers have now described the activity of p38 in cancer cells, but until now they did not know the key role that the protein plays in MSCs, and very little is known about how the protein is involved in tumor angiogenesis. In this study, researchers clarified the key role of the protein p38 in the cardiovascular process during tumor angiogenesis, especially how it promotes the development of MSCs. The researchers said that p38 can play a role in MSCs cells. The effect is to inhibit angiogenesis. Using genetically modified mouse models, researchers have found that inhibiting p38 may stimulate cardiovascular production in tumors, and this situation also occurs during the repair process of damaged tissues in the body. 3.EBioMedicine: researchers develop cancer treatments that target bone metastasis while retaining bone tissue doi: 10.1016 / j.ebiom.2019.06.047 Researchers at the University of California, Irvine (Irvine, UCI) have developed a treatment and tested it on mice using engineered stem cells to target and kill cancerous metastases in bone tissue while preserving bone. The new method, published in the journal EBioMedicine, equips engineered mesenchymal stem cells to target them, prompting them to transfer to bone metastases, where they release therapeutic drugs. "The power of this strategy is that we provide a combination of anti-tumor and anti-bone resorption agents so that we can effectively block the vicious circle between cancer and its bones," said the study's lead author Weian Zhao, associate professor of pharmaceutical sciences and biomedical engineering, said. "Compared to chemotherapy, this is a safe and almost non-toxic treatment, and chemotherapy often causes lifelong problems for patients." To be continued in Part Two…
  3. Exosomes are nano-scale vesicles secreted by cells. These microvesicles are usually about 30-150 nanometers in diameter and contain important cellular molecules such as proteins and RNA. Previous studies have shown that exosomes can be used as diagnostic markers for cancer, neurodegenerative disease, and kidney disease. In recent years, exosomes isolation technology has made significant progress and development. Ultracentrifugation Ultracentrifugation is the most commonly used exosomal purification method. After removing dead cells and cell debris by low speed centrifugation, high-speed centrifugation is used to precipitate vesicle particles of the same size from soluble molecules such as free proteins and protein complexes purified. It is important that the exosomes be subsequently washed at least once with PBS or fresh growth medium to reduce free residual proteins therein. In addition, all centrifugation steps must be performed at 4 ° C to keep the proteases, DNase and RNases inactive. Usually ultracentrifugation is also used in combination with a sucrose density gradient (its continuous distribution from low to high density) or a sucrose cushion (30% sucrose cushion), that is, centrifuged at 100,000-200,000 xg in a centrifuge (containing exosomes) In 120 minutes, the exosomes in the sample should be enriched in a sucrose density range of 1.13-1.19 g / mL. Although this powerful method can obtain highly purified exosomes, there are some disadvantages. Indeed, the process of ultracentrifugation is time-consuming and labor-intensive, and requires a lot of raw materials. The biggest drawback is that repeated centrifugation operations are likely to cause damage to exosomal vesicles and reduce their quality, or soluble proteins in the sample may form aggregates and clumps with exosomes to cause contamination. Ultrafiltration centrifugation Considering that exosomes are cystic bodies with a size of several tens of nanometers, which are larger than ordinary proteins, exosomes can also be separated according to their size, such as ultrafiltration and size exclusion chromatography (SEC). Ultrafiltration is the selective separation of samples using ultrafiltration membranes with different retention molecular weight (MWCO). That is, the solvent, ie, some small molecular substances, is filtered to the other side of the membrane, while high relative molecular mass substances larger than the membrane pore size are retained On the ultrafiltration membrane, the purpose of separating exosomes is achieved. This method is simple and efficient, and does not affect the biological activity of exosomes. It is the best method for studying exosomal RNA because it produces greater RNA production than ultrafiltration and precipitation methods. It is also possible to pass a nanofiltration concentrator. However, the main disadvantage of ultrafiltration is that exosomes may block the filter pores, resulting in shorter membrane life and lower separation efficiency. Exosome membranes also adhere to each other, resulting in low separation yields and even erroneous test results. In addition, there is another interference that needs to be resolved in the method of separating exosomes based on the size of the exosomes, which is the existence of a large number of non-exosomal nanovesicles that are similar in size to the exosomes. In SEC, the porous phase fixed in the column can also be selectively separated based on the molecular size using the principle of gravity flow. Small molecules can pass through the pores and cause later elution, while larger components (including exosomes) can be eluted early, bypassing the pores. This method can greatly maintain the integrity and biological activity of exosomes, and combine with differential centrifugation to obtain highly purified exosomes. PEG-base precipitation method Polyethylene glycol (PEG, 8000 kDa) can competitively bind free water molecules, so that less soluble molecules or exosomes are precipitated from the solution. Earlier this method was used to collect virus from samples such as serum, and now it is also used to precipitate exosomes. Samples are usually incubated overnight at 4 ° C with PEG, and exosomes are then recovered by low-speed centrifugation or filtration. However, this method also has some problems: for example, the purity and recovery of exosomes are low, false positives (more proteins or some polymers that are difficult to remove), and mechanical or chemical additives that damage the exosomes. Alternatively, if you know the sugar chain composition of the exosomes, you can use lectins to enrich the exosomes. Lectin is a protein that binds to carbohydrates and can be centrifuged at low speed after agglutinating exosomes. In recent years, exosomes have been separated based on the principle of precipitation. Various commercial exosomal extraction kits have also been developed on the market. The operation is simple, and high-purity and high-recovery exosomes can be obtained without ultracentrifugation. Magnetic bead immunoassay Exosomes are available because they are rich in protein and have many specific marker receptors on their surface, such as CD9, CD81, CD63, CD82, Hsp70, Ras-related protein Rab-5b, cytoskeleton protein actin, and TSG101. Anti-marker antibody-coated magnetic beads can be captured after incubation with exosomes. Because the heterogeneity of exosomes is consistent with their origin, the abundance of these markers on different exosomes is also different. Therefore, you can capture different types of exosomes from a sample by using specific antibody combinations, and select these exosomes by immobilizing these antibodies on ELISA plates, magnetic or chromatography beads, or microfluidic devices. Although immunoaffinity technology has the advantages of high specificity, high purity exosomes can be obtained without affecting the morphological integrity of exosomes, it is the preferred method for enriching and characterizing unique exosomes. However, this method is low in efficiency, and the biological activity of exosomal contents is easily affected by pH and salt concentration, which is not conducive to the downstream experiments. Phosphatidylserine affinity This method combines PS (phosphatidylserine) with magnetic beads and uses the principle of affinity to capture PS outside exosomal vesicles. This method is similar to the immunomagnetic bead method, and the exosomes obtained are complete in morphology and highest in purity. Since no denaturant is used and the biological activity of exosomes is not affected, exosomes can be used for cell co-culture and in vivo injection. 2016.9 "Scientific Reports" magazine published the latest data of this method, showing that PS method can extract relatively high purity exosomes. Chromatography The exosomes isolated by this method are uniform in size under electron microscopy, but require special equipment and are not widely used. Exosome isolation is the first step for exosome characterization. The quality of exosome separation directly affects the subsequent researches of exosome qualitative and quantitative as well as applications in disease diagnosis and therapy. With the extensive experience in exosome isolation, Creative Biolabs provides a portfolio of exosome isolation products which can help you with the high-quality exosome isolation from many types of biofluids in an efficient, faster and cheaper way.
  4. Vitamin E is a fat-soluble vitamin that was discovered as early as the 1920s. Vitamin E includes tocopherols and triene tocopherols, a total of 8 compounds. Alpha-tocopherol is the most widely distributed and most abundant form of vitamin E in nature. Tocopherol is a hydrolysis product of Vitamin E and is one of the most important antioxidants. How does Vitamin E help the human body? Vitamin E helps delay aging Vitamin E is a strong oxidant and is not weaker than lycopene and astaxanthin. After entering the body, vitamin E can help fight free radical Oxylipin peroxidation, eliminate free radicals, and delay aging. Vitamin E helps boost immunity If vitamin E is lacking, it will reduce the body's humoral immunity and cellular immunity, and increase the possibility of human diseases. Proper vitamin E supplementation will help to strengthen the body's ability to resist disease and enhance its physique. Vitamin E helps eliminate pigmentation Pigmentation is caused by the deposition of lipofuscin in skin cells. Lipofuscin is the product of cells being oxidized by free radicals. This substance not only produces stains and hinders aesthetic appearance, but also deposits in the internal organs and brain cells, causing cardiovascular and cerebrovascular diseases, and endangering health. Vitamin E as a strong oxidant can eliminate these free radicals, help prevent the generation of pigmentation, and at the same time tenderly expand peripheral blood vessels, reduce blood viscosity, and prevent cardiovascular and cerebrovascular diseases. Vitamin E can stabilize the protein active structure of the cell membrane, promote the normal development of muscles and maintain the elasticity of the skin, so that the skin and the body remain active; Vitamin E entering the skin cells can directly help the skin fight against the damage of free radicals, ultraviolet rays and pollutants, preventing The skin loses its elasticity due to some chronic or hidden injuries until it ages. Because of these effects of vitamin E, it is believed that vitamin E helps beauty. Vitamin E helps protect eyesight Vitamin E can inhibit the lipid peroxide response in the lens of the eye, expand the peripheral blood vessels, improve blood circulation, and prevent the occurrence and development of myopia. Vitamin E helps relieve stomach ulcers The poor gastric mucosal resistance in patients with ulcer disease is related to the disturbance of fat peroxidation. Vitamin E can regulate fat oxidation and scavenge oxidative free radicals, while protecting cells from oxidant damage. At the same time, a large amount of vitamin E can promote the proliferation of capillaries and small blood vessels, improve the surrounding blood circulation, increase the supply of oxygen in the tissue, thereby creating good nutritional conditions for healing of the ulcer surface. In addition, it can still inhibit the growth of H. pylori and reduce the recurrence rate of ulcer disease after healing. Vitamin E helps promote sex hormone secretion Vitamin E can increase men's sperm vitality and quantity; increase women's estrogen concentration, improve fertility, and prevent miscarriage. Is it okay to take a lot of vitamin E for a long time? Vitamin E is found in edible oils, fruits, vegetables and grains. The recommended daily intake for adults is 8 to 10 IU. Vitamin E in the general diet can completely meet the needs of the human body. Therefore, the general population does not need to take vitamin E for a long time. Long-term use is not only unsafe, but also has side effects. Taking large doses of vitamin E for a long time may cause various diseases. The more serious ones are: Intake of low-dose vitamin E has anti-oxidant effect, but it may no longer have antioxidant activity when ingested in large doses. At this time, vitamin E becomes a pro-oxidant; Thrombophlebitis or pulmonary embolism, or both, is due to the high dose of vitamin E that can cause platelet aggregation and formation, which may trigger the risk of stroke; Headache, dizziness, dizziness, blurred vision, muscle weakness; skin cracking, cheilitis, angular cheilitis, urticaria;
  5. In recent years, studies have found that the incidence of primary malignant brain tumors has increased significantly, and the current prognosis is poor. Therefore, studying the mechanism of brain tumor recurrence, improving the prognosis of patients, and prolonging their survival time is an important research direction and a major problem faced by experts: due to the vague understanding of the source of brain tumor cells and its mechanism. Although a large number of related experimental studies have been done on the pathogenesis of malignant brain tumors, but no satisfactory results have been achieved. Currently, only a small percentage of cells in surgical tissues of brain tumors have been found to have infinite proliferation, self-renewal, multi-directional differentiation potential, and tumorigenicity. These cells are called brain tumor stem cells (BTSCs), and others tumor cells have no or only short-term proliferation ability. Igntova and other scholars first reported that brain tumor stem cells (BTSC) existed in brain tumor surgical specimens, and isolated precursor neurons that can form neurospheres from glioblastomas, which are called neural stem cells in brain tumors. At present, each brain tumor stem cell has been successfully cultured and isolated from surgical specimens such as medulloblastoma, different grades of astrocytoma, ependymal tumor, and ganglioglioma. Since Singh et al. First isolated CD133-positive tumor stem cells from malignant brain tumors, research on brain tumor stem cells has gradually become a hot topic in neuroscience and related fields. Preliminary studies have found that the occurrence, development, metastasis, and recurrence of brain tumors may be closely related to brain tumor stem cells. Therefore, further in-depth discussion of the biological characteristics of brain tumor stem cells and the mechanism in the occurrence and recurrence of malignant brain tumors will definitely be very important for the future of radical treatment and prevention of malignant brain tumors. Here in this article, we will introduce three important biomarkers. One of the most prominent one is TSGF, namely a group of tumor-related substances. It is a collective term for several internationally recognized carbohydrates and metabolites (lipoproteins, enzymes, amino acids) related to the growth of malignant tumors. TSGF is an effective, convenient and valuable tumor marker for the diagnosis and judgment of brain malignant tumors. In addition, studies have shown that the expression specificity of tumor markers such as nestin, BEHAB, YKL-40, EphA2, glial fibrillary acidic protein, CD133, fatty acid binding protein, and MMP-9 is more obvious in gliomas. 1. BTSC There are many hypotheses about the source of BTSC, but currently they tend to be derived from mature neural stem cells (NSCs). The accumulation of multiple mutations leads to tumorigenicity and becomes BTSC. The source is discussed from the following two aspects: ① The origin of the tumor is consistent with the NSC distribution area. Studies have shown that the origin of brain tumors may originate in a part of the subventricular area, and BTSCs with high proliferation and differentiation potential are constantly produced in this area, which leads to tumorigenesis, and these areas coincide with the main locations of NSCs. ② BTSC and NSC have many similarities in genetics. The main manifestation is that BTSC does not express markers of differentiated cells, but instead has NSC markers, such as Nestin or CD133. In summary, both theory and experiments support that BTSC is likely to be derived from mutant NSCs that are constantly dividing and proliferating. Although BTSC and NSC have many similarities, there are also obvious differences between them: first, BTSC has stronger self-renewal and proliferation capabilities than NSC, and the number of passages in vitro culture has increased significantly, with an immortalization trend. Its self-renewal and differentiation have become imbalanced; secondly, BTSC differentiates into the same phenotype as the parent tumor under the conditions that induce NSC differentiation and does not differentiate into neurons and glial cells in the same proportion as NSC. These differences provide new research directions and ideas on how to transform NSC into BTSC and whether the two are at the same level of differentiation. 2. CD133 protein, nestin, Sox2 protein In recent years, Rath et al. have successfully cultured and isolated meningioma stem cell spheres with spherical focus growth through serum-free suspension culture. At present, most scholars use CD133 and Nestin as specific markers of brain tumor stem cells. CD133 is a transmembrane protein with a relative molecular weight of 120,000. Studies have shown that both solid tumors and brain tumor cell spheres obtained from in vitro cell line cultures show CD133 positive staining; and CD133 positive cells isolated from glioma cell lines in serum-free culture, they all grew spheroidally, had infinite proliferation, self-renewal, and multi-directional differentiation. Singh et al. compared the biological characteristics of CD133-positive and negative tumor cells and found that the former has a strong ability to self-renew and proliferate, while the latter adheres to growth, does not divide, and does not proliferate. In vivo tumorigenicity tests showed that 100 CD133-positive tumor cells were tumorigenic, while 1 × 105 CD133-negative tumor cells formed only one glial scar at the transplant site. For these reasons, CD133 is considered to be the most important marker of brain tumor stem cells. However, recent studies have shown that CD133-negative cells in some brain tumors also have the characteristics of tumor stem cells. Therefore, CD133 is not a reliable marker for brain tumor stem cells. Nestin, which belongs to the intermediate microfilament, is expressed in undifferentiated neural pluripotent stem cells and was once considered a marker of brain tumor stem cells. However, the study found that the same group of tumor cells, Nestin-positive ratio is much higher than CD133-positive ratio, which indicates that Nestin is also expressed in progenitor cells that have just begun to differentiate, and is not a reliable brain tumor stem cell marker. Sox-2 belongs to the Sox (Sry-related HMG Box) gene family and is located at 3q26.3-q27 of the chromosome. It is a highly conserved transcription factor that can regulate the self-renewal of embryonic stem cells. It is the only Sox gene found in current research that plays an important role in maintaining the differentiation potential of embryonic stem cells. It is also a key to induce adult cells to become pluripotent stem cells. 3. Brain tumor stem cells The research of brain tumor stem cells has become a new hot spot in the field of brain tumor research. Although great achievements have been made in the successful isolation and culture of brain tumor stem cells, no complete theoretical system has been formed so far. Therefore, it is of great significance to study the pathogenesis and biological behavior of brain tumors and to find new treatment options for malignant brain tumors that target tumor stem cells in the future. It can be imagined that the detection of tumor stem cells will become a new classification and judgment of brain tumors in the future. It is possible to use various treatment schemes for brain tumor stem cells to specifically kill brain tumor stem cells, instead of killing all tumor tissues, in order to achieve radical cure and prevent tumor recurrence and metastasis. With the continuous research on brain tumor stem cells, it will inevitably have a profound impact on the pathogenesis, pathological grading, prevention of recurrence and treatment options of brain tumors, making the radical cure of malignant brain tumors possible.
  6. According to previous data, CD19 was a hot CAR-T treatment target, and has achieved great success in relapsed and refractory B-cell malignant blood diseases, and there are already two commercial products for the treatment of relapse fefractory B-cell acute lymphocytic leukemia (B-ALL) and relapsed refractory non-Hodgkin's lymphoma (NHL). But not all patients benefit from CD19 CAR-T therapy. For example, most clinical data reports indicate that CD19 CAR-T has an objective response rate (ORR) of 80% and a complete response rate (CR) of approximately 50% in patients with relapsed and refractory NHL, and nearly 20% of non-responses and more than half of the patients fail to achieve sustained remission. Why can't some non-Hodgkin's lymphoma patients benefit from it? Why is it? Because the CD19 antigen is lost on the cancer cells of these patients, for these patients, CD19 CAR-T no longer has the ability to target and attack cancer cells, which in turn can lead to the recurrence of cancer. How to solve this problem? CAR-T therapy targeting CD20 may be an alternative solution. CD20 is a human B lymphocyte restriction differentiation antigen, encoded by the MS4A1 gene (located at 11q12). This antigen is a hydrophobic 4-pass transmembrane protein with a molecular weight of approximately 35 kD, Leukocyte surface antigen Leu-16, transmembrane 4 domain subfamily A member 1 and so on. This protein function may be involved in regulating B cell activation and proliferation, and may function as a calcium ion channel. CD20 antigen is mainly present on pre-B and mature B lymphocytes, and is expressed on most B-cell non-Hodgkin lymphoma cells, but not on stem cells, pro-B cells, normal plasma cells, or other normal organizations. Plasma cells naive and stimulated plasma cells may express CD20. According to the expression characteristics of CD20 in the development stage of B lymphocytes, it has been selected as one of the targets for the treatment of B-cell lymphoma and leukemia, and many antibody drugs have been successfully developed based on this target. Research progress of CD20 CAR-T therapy 1. In China As early as the end of 2012, the Molecular Immunology Department / Biotherapy Ward of the General Hospital of the Chinese People's Liberation Army launched a clinical trial of CD20 as a target for CAR-T cell therapy for relapsed and refractory diffuse large B-cell lymphoma (DLBCL). The study, completed in 2014 and first reported the results of a phase I clinical study in 7 patients, showed that CD20 CAR-T cells combined with a tumor-reducing pretreatment regimen can prolong tumor regression (Wang Y, et al. ClinImmunol. 2014). At the same time, the team also applied to the State Intellectual Property Office for a CD20 CAR-NKT patent (engineered CD20-targeted NKT cells and its preparation method and application, patent application number: 201410062069.7). At the beginning of 2015, Sibeman chose to cooperate with Han Weidong, director of the Molecular Immunology Laboratory of the Life Sciences Institute of the General Hospital of the PLA, to help Professor Han Weidong develop CD20 as a representative by leveraging his successful experience in translational medicine in the biomedical field and sufficient R & D funding advantages. Based on the phase I clinical research, Han Weidong's team launched a phase IIa clinical study of CD20 CAR-T in the treatment of relapsed and refractory NHL. The results of related studies in 11 patients were included and the results were published in "Signal Transduction and Targeted Therapy" in October 2016. Six of the 11 patients were evaluated as CR after CAR-T infusion (1 of whom was transferred from Phase 1 to Phase IIa and continued CD20 CAR-T alone), 3 were PR, and 2 were stable (SD ), 2 patients (1 PR, 1 SD) also received CR after local radiotherapy in the later stage. In early October 2017, the Han Weidong team published a research report again in the "Signal Transduction and Targeted Therapy". Based on a retrospective review of the clinical outcomes of 16 patients who can be evaluated after the test, the article highlights that 8 patients achieved CR after CAR-T infusion (or combined local radiotherapy), with the exception of 3 patients who had recurrence. As of the end of July 2017, 5 patients were still in the state of continuous CR (1 from the stage I subjects and 4 from the stage IIa subjects), of which 1 patient continued the CR stage and it has lasted 57 months, 3 cases exceeded 40 months, and 1 case exceeded 20 months. At the same time, in July 2017, the NCI in the United States reported the long-term follow-up results of CD19-CAR-T in the treatment of NHL in Molecular Therapy, showing 5 of 7 patients with CR, 4 of whom had a continuous CR period of 56, 51, 44, 38 months. These two studies show that CAR-T treatment can not only achieve short-term efficacy in patients with relapsed and refractory NHL, but also can obtain long-term CR efficacy in most patients who obtain CR after CAR-T. It also indicates the long-term effectiveness of CAR-T therapy for CD20 in patients with relapsed and refractory NHL. In terms of long-term safety, Han Weidong's team also observed that patients with continuous CR after CAR-T treatment are often accompanied by longer-term B cell deficiency or low, and low immunoglobulinemia. Of the 5 long-term CR patients, except for one patient who developed grade 3 shingles infection in July after CAR-T treatment, the remaining patients can effectively prevent the occurrence of grade 3 infectious diseases through regular supplementation of gamma globulin . 2. In US In September 2017, Fred Hutch, a top cancer center, licensed a CD20 CAR-T therapy developed by Mustang Bio to the clinic as soon as possible. Phase I / II clinical trials with partial support from Mustang Bio will be led by Dr. Mazyar Shadman, Clinical Research, Fred Hutch. The trial will recruit approximately 30 patients with relapsed or refractory B-cell non-Hodgkin's lymphoma (B-NHL). Eligible patients will first undergo a biopsy to ensure that their tumors have a CD20 marker. The researchers expressed hope that as shown in preclinical studies, CD20 CAR-T therapy could even be more effective than CD19 CAR-T.
  7. Co-immunoprecipitation (Co-IP) is a classical method for studying protein-protein interactions based on the specific role of antigens and antibodies. The development of co-immunoprecipitation technology has gone through several stages. In the early days, researchers used gel electrophoresis to isolate co-immunoprecipitated proteins: the antigen solution was added to small wells such as agarose, and antiserum was added to adjacent wells. With the antigen and antibody diffusing, large molecules entered in the gel, and an interaction occurs between the two to form a complex. A concentration gradient is formed by diffusion of the antigen and antibody, and a multi-molecular network complex is formed at the optimal concentration. Finally, the large-molecular protein complex is precipitated from the solution. With the development of society, the co-immunoprecipitation technology has been continuously improved. The method of isolating the immune co-precipitation complex is improved to promote the multimer reaction, so that the immune complex is precipitated from the solution. In the mid-1970s, people began to use solid-phase reactions, using protein A immobilized on the surface of S. aureus to adsorb antibodies, and then bind to the corresponding antigens. With the development of science and technology, this method has been improved to use the surface-immobilized protein A or protein G microspheres to separate antigen-antibody complexes in order to achieve the purpose of detecting antigens or target proteins. Principle of co-immunoprecipitation The experimental principle of co-immunoprecipitation is: if X is immunoprecipitated with an antibody to protein X, the protein Y bound to X in the body can also be precipitated. At present, prorein A is often pre-bound and solidified on agarose microbeads and reacted with the solution containing the antigen and the antibody. Prorein A on the agarose microbead specifically binds to the antibody Fc. Due to the specificity of the antibody antigen, protein X is precipitated; There are substances that interact with protein X, and they can precipitate. Application of co-immunoprecipitation Determine whether two target proteins are bound in the body Identify a new role for a particular protein Isolate and obtain the interaction protein complex in its natural state With the continuous deepening of protein research, people have combined immunoprecipitation methods with other methods, and based on them, many more complicated technologies have been derived, which makes the analysis methods more diverse and its application range is quite wide. Co-immunoprecipitation is a technique used to study protein-protein interactions and can be applied to the study of protein complexes. It can verify the existence of protein complexes, and then discover new protein complexes. Co-immunoprecipitation technology is combined with immunoblotting or mass spectrometry to determine the binding of bait protein-target protein in its natural state and specific proteins. Co-immunoprecipitation experiments can also be applied to the enrichment and concentration of low-abundance proteins. At the same time, co-immunoprecipitation is a relatively classic technique for exploring protein-protein interactions. It has a wide range of applications and high credibility in modern medical research. Protein interactions permeate the life activities of every cell in the body. Many phenomena in biology such as replication, transcription, translation, shearing, secretion, cell cycle regulation, signal transmission and intermediate metabolism are all affected by proteins. Some proteins are composed of multiple subunits, and protein-protein interactions are particularly prevalent. Some proteins bind very tightly, while others interact only briefly. However, no matter what kind of situation occurs, they control a large number of events of cell life activity, such as cell proliferation, differentiation and death. And through protein-protein interactions, it can change the dynamic characteristics of intracellular proteins, such as substrate binding properties and catalytic activity. It can also generate new binding sites, which has an effect on changing the specificity of proteins on substrates. Therefore, only by allowing the interaction between proteins to proceed smoothly, the normal life activities of cells can be guaranteed. Because protein-protein interactions are so significant, the study of their detection methods has also received much attention. Since then, the research on protein interrelationships will intensify. In the future, it will not only be confirmed by co-immunoprecipitation technology, but more and more advanced technologies will be worth applying and developing. The pros and cos Advantage: The protein exists in a natural state after modification and translation; Detects interactions in vitro and in cells; Antigens and interacting proteins are present at similar concentrations in cells, avoiding human effects caused by overexpression; It is possible to isolate the interacting protein complex in its natural state. Disadvantages: The sensitivity is not as high as that of affinity chromatography; High false positive rate, correct control is necessary; The binding of two proteins may not be directly combined, but a third party may act as a bridge in the middle; It is necessary to predict what the target protein is before the experiment in order to select the antibody to be detected at last, so if the prediction is incorrect, the experiment will not yield results, and the method itself is risky; After the protein forms a complex, some epitopes will be masked, which may lead to the use of a pull-down antibody. No matter how the antibody concentration is increased, less than half of the target protein complex can be precipitated. If necessary, it is best to use multiple different antibodies for Co-IP respectively; Since the natural state is detected, the protein complexes pulled down by Co-IP may be different under different times and different treatments. Of course, as the number of experiments increases, the members of the obtained protein complexes will also bedifferent. Authenticity of co-immunoprecipitation results To ensure the authenticity of experimental results in co-immunoprecipitation experiments, the following points should be noted: Make sure that the co-precipitated protein is obtained by precipitation of the added antibody, not a non-exogenous non-specific protein. Monoclonal antibodies have the advantages of strong specificity, mass production, and easy standardization. The use of monoclonal antibodies can help avoid contamination; To ensure the specificity of the antibody, if the antibody cannot bind to the antigen in the cell lysate, it will not cause a co-precipitation reaction; Make sure that protein-protein interactions occur in cells, not because of lysis of the cells.
  8. 3. Optimal dosage The optimal dose of MSC depends on the different disease and severity and the route of entry. In the clinical research and application of MSC, cell dose may belong to the most clueless and most scientific aspect. Even if there are some clinical studies involving dose climbing experiments, they are not based on animal experiments. MSC is different from traditional medicines, because: first, after MSC enters the body, it does not conform to the typical distribution and metabolism model of traditional medicines; traditional medicines are passive distribution, and MSCs have the function of actively chemotactic to the injury site, and the distribution of MSCs in healthy and diseased organisms is different. Second, animal experiments of traditional medicines require multiple administrations to maintain a stable blood concentration, and animal experiments of MSCs are often single injections, so that clinical studies of MSCs often adopt a single injection scheme. In fact, a single injection of MSC cannot achieve a good stable long-term treatment effect, even if there is a significant improvement in the short term. For a certain disease, in the preclinical studies, animal experiments have not fully demonstrated the minimum and maximum saturation doses at which MSCs work, and the cell doses in different laboratories differ. Factors such as the culture system of different laboratories and the source properties of MSC often lead to differences in the quality of MSC, which directly affects the results of animal experiments and clinical studies of MSC. Therefore, data from preclinical studies of MSCs do not provide a good guide for determining clinical research protocols. In current clinical studies, the dosage range of MSC used is very large, and the number of MSC cells used per patient ranges from more than 4,000 MSCs to hundreds of millions of MSCs. For local intervention, the lowest dose appears in the clinical case of MSCs for femoral head necrosis. Each clinical study in Korea and France used more than 4,500 MSCs. The highest dose of interventional therapy appeared in a clinical study in China, which used 860 million cells for MSCs to treat diabetic limb bullae; the second highest dose was 200 million MSC myocardial injections. Clinical studies of more than 100 million MSCs injected locally include: 120 million MSCs for Crohn's disease intestinal fistula, 100 million MSCs for intraarticular injection of knee osteoarthritis, and 100 million MSCs for ischemic cardiomyopathy. Intravenous doses of cells are relatively stable, and millions of MSCs per kilogram of body weight are often used, ie (1-10) x 10 * 6 / kg. The highest intravenous dose is 10x10 * 6 / kg co-transplanted with hematopoietic stem cells. According to a weight of 60 kg, that also requires 600 million MSCs; and 8x10 * 6 / kg for GVHD. The lowest dose of intravenous injection appeared in clinical trials of MSC and hematopoietic stem cell co-transplantation, which was 0.3x10 * 5 / kg. The failure of the Phase 3 clinical trial of Prochymal (bone marrow MSC) in the treatment of refractory GVHD in 2009 was a catastrophic event in the clinical application of MSC, which almost denied the clinical efficacy of MSC. Prochymal is derived from the bone marrow of healthy people, and MSC itself has a strong immunosuppressive ability. However, why can't Prochymal be significantly better than the control group in the phase 3 clinical trial? At that time, Prochymal's indication was hormone-resistant and refractory GVHD. This indication itself was very difficult. If Prochymal did not optimize the treatment plan, especially the breakthrough in conventional thinking in terms of dosage, then failure would be inevitable. Later increasing the dose of MSC cells to 5x10 * 6 / kg and 8x10 * 6 / kg, the effect of treating hormone-resistant refractory GVHD was better than that before 2009, making the UK and EU treatment guidelines recommend MSC as a treatment for grade 2-4 Third-line treatment for acute GVHD. But Prochymal is still not approved by the US FDA. Interestingly, a meta-analysis article in 2016 suggested that the "dose" factor did not affect the survival rate of MSC in patients with acute GvHD. Although some experts believe that 5x10 * 6 / kg and 8x10 * 6 / kg are high doses, there is no discussion and proof of what kind of dose is defined as "high dose". If MSC is regarded as a "medicine", then there must be a range. In this range, the higher the dose, the better the effect; then after reaching a saturated dose, continuing to increase the cell dose does not bring more efficacy, but may bring some adverse reactions. To be continued in Part Four…
  9. Immune oncology (IO) is the focus of current drug development. In recent years, the development of many IO drugs around the world has been rapidly improving not only in speed but also in quality, which brings new hope to almost all types of cancer patients, including many rare cancer types. At present, IO therapy targeting PD- (L) 1 has achieved great clinical success, and has attracted more and more research teams. Many agencies have predicted that in the next few years, the size of the global IO market will continue to grow rapidly. According to GrandView Research, the size of the IO market in 2018 was $ 58.1 billion, and it is expected to reach $ 126.9 billion in 2026. Transparency Market Research and Market Research Engine predict that the IO market will reach 124.88 billion US dollars and 173 billion US dollars in 2024, respectively. These optimistic predictions showed a significant increase in clinical activity, with the expected launch of many new treatments for IO. Last year, Incyte and Merck Epacadostat / Keytruda encountered huge failure in the clinical phase III of melanoma. This hit the development of epacadostat and other IDO inhibitors. At the same time, it also reduced the industry's enthusiasm for development of cancer immunotherapy. However, research on novel immune checkpoint targets that stop tumor cells from fighting the immune response or stimulate the body's immune system against cancer is continuing. A recent statistics from the American Cancer Institute (CRI) found that more than 1,700 joint studies involving PD- (L) 1 inhibitors are currently underway. Early and mid-term research data on these novel immune checkpoint targets may be seen at the 2019 American Society of Clinical Oncology (ASCO) Annual Meeting. 1.Target: STING (interferon gene complex stimulant) Type: Immune stimulation Related companies: Aduro and Novartis' ADU-S100, GlaxoSmithKline's GSK3745417, Merck MK-1454 Aduro and Merck's STING pathway candidate drugs are administered by intratumoral injection, meaning they can be used for melanoma, but may not have much applicability for cancers deep in the body. The MK-1454 data released at the 2018 European Society of Oncology Medical Association showed that the efficacy of combination with Keytruda was not obvious. GSK3745417 from GlaxoSmithKline (GSK) can be administered by intravenous infusion, and early drug trials are currently underway. Data from the combined use of ADU-S100 and Novartis anti-PD-1 therapy spartalizumab will be presented at this year's ASCO Annual Meeting. 2.Target: LAG-3 (lymphocyte activating gene-3) Type: Immunosuppression Related companies: GSK TSR-033, Regenerative REGN 3767; Bristol-Myers Squibb BMS-986016, F-Star Corporation FS118 TSR-033 is one of GSK's $ 5.1 billion acquisition of Tesaro's oncology assets. The clinical progress of the drug has received much attention. Regeneron and BMS will also show the latest progress of their drug candidates and their respective PD-1 inhibitors at the ASCO Annual Meeting. F-Star's FS118 is a bispecific antibody that targets both PD-1 and LAG-3. The drug has signed an option agreement with Merck, but recently Merck abandoned its option and F-Star re- Obtained full development rights for the drug. 3.Target: TIM-3 (T cell immunoglobulin and mucin domain-3) Type: Immunosuppression Related companies: Eli Lilly LY3321367, BeiGene BGB-A425, Symphogen SYM023, Novartis MBG453 This class of drugs is a classic match with PD- (L) 1. All of the above drugs are in Phase I clinical trials, in combination with experimental PD- (L) 1 drugs from each company and with externally approved immune checkpoint inhibitors. Early safety data provided by Eli Lilly showed high levels of anti-drug antibody response (without affecting pharmacokinetics) and evidence of tumor response. At present, no other data on this class of drugs has been released. 4.Target: TGFβ Type: Immunosuppression Related companies: Merck and GSK's bintrafusp alfa (bispecific PD-L1), Forbius Avid200, Lilly Galunisertib, University of Pennsylvania CART-PSMA-TGFβRd. Of all the oncology challenges, the biggest bet may be Merck's decision to confront Merck in frontline non-small cell lung cancer. The hypothesis of this study is that bispecific bintrafusp alfa can block the PD-1 pathway as effectively as Keytruda, while inhibiting TGF-β provides additional benefits by blocking another mechanism that tumors use to shut down the immune response. Meanwhile, Eli Lilly has been evaluating galunisertib for the past few years and has reported some promising phase II data for pancreatic cancer. TGFβ also appears to be the only novel immune checkpoint target on the list considered a potential therapeutic technology, and the University of Pennsylvania is currently testing metastatic castration resistance to prostate cancer. 5.Targets: OX40 Type: Immune stimulation Related companies: Pfizer PF-04518600, Bristol-Myers Squibb BMS986178, Incygn IncAGN1949 These drug candidates are in very early human clinical trials testing dose levels and biological responses. BMS research combined BMS986178 with Opdivo and Yervoy and found that BMS986178 stimulated the immune response. Pfizer's PF-04518600 has achieved disease stabilization in some patients. Creative Bioarray possesses a research team, which has been focused on Immune-Oncology for decades. Since there are still lots of incomplete scientific understanding of tumor immunology, which gives rise to little widespread use of I-O therapies in clinic, Creative Bioarray is always ready to help and cooperate with customers on their I-O research or preclinical experiment. With multitudinous tumor cells, diverse animal tumor models, and various tumor research related services, such as cell cycle assays, apoptosis assays, proliferation assays, migration assays and toxicology assays, Creative Bioarray could offer professional design and experiment for customers to facilitate their I-O program.
  10. The effect of denaturing enzyme protein and causing loss of enzyme activity is called enzyme inactivation. The effect of reducing enzyme activity without causing enzyme protein denaturation is called inhibition. Enzyme inhibitory effects include irreversible inhibition and reversible inhibition. Some substances do not cause the enzyme protein to denature, but they can change certain essential groups (some groups on the active center) on the enzyme molecule, causing the enzyme activity to decline or even be lost, which are called enzyme inhibitors. Scientists can use inhibitors to study enzymes; drug manufacturers can use inhibitors as drugs to treat diseases; and inhibitors can also be toxic. Here in this article, enzyme inhibition are mainly discussed. Enzyme inhibition refers to the effect that the functional group of an enzyme is affected by a certain substance, resulting in a decrease or loss of enzyme activity. This substance is called an enzyme inhibitor. Enzyme inhibitors are selective for enzymes and are an important tool for studying the mechanism of enzyme action. Many drugs, poisons and poisons used in chemical warfare are enzyme inhibitors. In addition, some biological macromolecules that exist in animals and plants with certain functions are also enzyme inhibitors. When the enzyme is inhibited, its protein portion is not denatured. Enzyme inactivation due to enzyme protein denaturation, and the reduction or loss of enzyme activity caused by the removal of activators (such as metal ions necessary for enzyme activity) are not included in the scope of enzyme inhibition. Inhibitors can be divided into reversible inhibitors and irreversible inhibitors. Irreversible inhibitor Irreversible inhibitors are mainly covalently bound to enzymes, reducing enzyme activity. Covalent binding is tightly bound, and physical effects such as simple dialysis and dilution cannot be used to remove the inhibitory effect. Reversible inhibitor Reversible inhibitors bind through non-covalent bonds and have weak binding power, so they can both bind and dissociate easily, and quickly reach equilibrium. Reversible inhibitors are divided into two categories: competitive inhibitors and non-competitive inhibitors. (1) The structure of competitive inhibitors is similar to that of substrates. It mainly binds to the binding groups of essential groups and competes with substrates for enzymes. The ability of the inhibitor to compete with the substrate for the enzyme's binding site depends on the concentration of both. If the inhibitor concentration is constant and the substrate concentration is low, the inhibitory effect is most obvious. As the substrate concentration increased, the enzyme-substrate complex concentration increased, and the inhibitory effect weakened. When the substrate concentration is much higher than the inhibitor concentration, almost all enzymes are taken by the substrate. At this time, the Vmax of the enzymatic reaction remains unchanged, but the Km value becomes larger. Many drugs are competitive inhibitors of enzymes. Sulfa drugs have a similar structure to para-aminobenzoic acid, which is a substrate for dihydrofolate synthetase. Therefore, sulfa drugs competitively inhibit dihydrofolate synthase, causing bacteria to lack dihydrofolate or even tetrahydrofolate. Inability to synthesize nucleic acids and inhibit proliferation. (2) The combination of non-competitive inhibitors with sites outside the active center of the enzyme does not affect the binding of the enzyme to the substrate, and the substrate does not affect the binding of the enzyme to the inhibitor. There is no competitive relationship between the substrate and the inhibitor. However, the substrate-enzyme-inhibitor complex cannot further release the product, so it is called non-competitive inhibitory effect, which shows a decrease in Vmax value and constant Km value. (3) Anti-competitive inhibitors only bind to enzyme-substrate complexes, reducing the amount of intermediate products. The significance of studying enzyme inhibitors Helps to study the catalytic mechanism of enzymes and the design and development of inhibitor-type drugs,such as anticancer drugs; Artificially regulate the metabolic pathways of the organism; It is helpful to study the relationship between the structure and function of enzymes. Creative Enzymes gladly supply various enzyme inhibitors of premier grade to the customers. We persist in being the most reliable supplier for enzyme products in the global market. Today, Creative Enzymes is a leading company in enzymes and enzyme-related products, and is well known for the high level of customer satisfaction. We deliver the products in a momentary span of time from order placement to final delivery. Our prompt service, dedicated customer care, and reliable approaches have made us the most preferred vendor.
  11. Biological or chemical substances originally added or artificially added to foods that have acute or chronic hazards to human health is the so called food contamination. Classification of food contamination According to the nature of the source, food contaminationis divided into biological contamination, chemical contaminationand radioactive contamination. (1) Biological contamination of food includes microorganisms, parasites and insects, toxic biological tissues, and insects. The main contamination is microbial contamination, which is more harmful, especially bacteria and bacterial toxins, mold and mold toxin. (2) The source of chemical contamination is complex and various. For example, ① Pollutants from production, life and environment, such as pesticides, harmful metals, polycyclic aromatic hydrocarbon compounds, N-nitroso compounds, dioxins, etc. ② From the production, processing, transportation, storage and sales of tools, containers, packaging materials and coatings and other materials into the food materials, monomers and additives and other substances. ③ Substances generated during food processing and storage, such as harmful alcohols and aldehydes in alcohol. ④ Abuse of food additives. (3) Radioactive contamination: the man-made radionuclide contamination in the environment mainly comes from the following aspects: nuclear explosion, discharge of nuclear waste, accidents. Radionuclides in the environment can be transferred to food through the food chain. The main transfer pathways are: transfer to aquatic organisms, to plants, and to animals. Harm to human body caused by radioactive contamination of food include long-term irradiation effects of low-dose radiation on various tissues, organs and cells in the human body after ingestion of contaminated food. It shows damage to the immune system and reproductive system, as well as carcinogenic, teratogenic and mutagenic effects. According to the way of food contamination, it is classifiedas: 1). Endogenous contamination (1) Endogenous contamination (2) Endogenous biological contamination: Livestock and poultry are infected with zoonotic diseases (meat, eggs, and milk are contaminated); Livestock and poultry are infected with inherent diseases, and their resistance decreases to cause secondary infections. During the life of livestock and poultry, some microorganisms are contaminated, and the decline in the resistance of livestock and poultry causes these microorganisms to infiltrate into muscle, liver and other parts, causing meat contamination. (3) Endogenous chemical contamination (4) Endogenous radioactive contamination 2). Exogenous contamination Exogenous biological contamination: food processing, transportation, storage, sales, cooking and other processes due to non-compliance with operating procedures, resulting in contamination by microorganisms, etc. (1) through water contamination (2) through air contamination (3) Contamination through soil (4) Contamination during production and processing (5) Contamination during transportation / storage (6) Contamination by vector pests Exogenous chemical contamination: Food is contaminated by toxic chemicals during processing, transportation, storage, sales, cooking, etc. The affected link include: (1) air (2) water (3) soil 4) transportation (5) production and processing Characteristics of food contamination Food is becoming more and more contaminated, and chemical contamination is the main cause. When pollutants are transferred from one organism to another, the concentration can be continuously accumulated and increased, which is the so-called bio-accumulation effect, and even the slight contamination process can cause serious harm to the human body after bio-accumulation. The hazards caused by food contamination today are more common than chronic toxicity, in addition to acute toxicity. Due to a small amount of human exposure for a long time and a long biological half-life, food contaminants have played a role in DNA in the body. The harm of food contamination to the human body Affect the sensory characteristics of food; Cause acute food poisoning; Cause acute and chronic hazards to the body; Teratogenic, mutagenic and carcinogenic effects on humans. The hazards of biological contamination: cause animal food corruption and deterioration; human infectious diseases; microbial poisoning. Harm of chemical contamination: acute, chronic poisoning; mutagenicity; teratogenic and carcinogenic.
  12. Common reasons for the temperature rise of TPE injection molding machines include the following: (1) The volume of the oil tank of the injection molding machine is too small, the heat dissipation area is insufficient, and the cooling device has a small capacity. (2) The fixed pump oil supply system that selects the oil pump capacity according to the fast forward speed, the excess flow will return from the relief valve under high pressure and generate heat during work. (3) The unloading circuit in the system fails or because the unloading circuit is not set, the oil pump cannot be unloaded when it stops working, and the entire flow of the pump overflows under high pressure. Overflow loss occurs and heat is generated, resulting in excessive temperature. (4) The system piping is too thin and long, too much bending, local pressure loss and pressure loss along the process are large. (5) Insufficient component accuracy and poor assembly quality, and large mechanical friction loss between relative movements. (6) The mating clearance of the mating parts is too small, or the gap is too large after use, the internal and external leakage is large, and the volume is lost. For example, the volumetric efficiency of the pump is reduced, and the temperature rises quickly. (7) The working pressure of the hydraulic system is adjusted much higher than the actual need. Sometimes it is because the seal is too tight, or because the seal is damaged, and the leakage increases, it is necessary to increase the pressure to work. (8) High climatic and operating environment temperatures cause the oil temperature to rise. (9) The viscosity of the oil is not selected properly. If the viscosity is too large or too small, it can cause heat generation and the temperature is too high. Approach: 1. According to different load requirements, check and adjust the pressure of the relief valve from time to time. 2. Reasonably select hydraulic oil, especially the viscosity of the oil. When the conditions allow, use a lower viscosity to reduce the loss caused by viscosity friction. 3. Improve and improve the lubrication conditions of moving parts to reduce friction loss, which is conducive to reducing work load and reducing heat generation. 4. Improve the assembly quality and accuracy of hydraulic components and hydraulic systems, strictly control the clearance of the mating parts and improve the lubrication conditions, use sealing materials with low friction coefficient and improve the sealing structure, and reduce the starting force of the hydraulic cylinder as much as possible Heat generated by mechanical friction losses. Add cooling device if necessary
  13. Rinderpest virus Rinderpest is an acute subacute infectious disease that mainly infects ruminants, especially cattle. It is characterized by severe hemorrhagic catarrh in the mucosa, accompanied by necrotizing stomatitis and gastroenteritis, with low congenital resistance. The mortality rate of animals is high. Rinderpest originated in Asia and was introduced into Europe, causing serious losses. At present, rinderpest has been eliminated in Europe and the United States, and China has declared it out as early as the 1950s. However, the disease is still prevalent in Equatorial Africa and Northeast Africa. And this disease is also widespread in Afghanistan, Pakistan, Southeast Asia and other places bordering China. Physicochemical properties The rinderpest virus is considered to be weakly resistant to physical and chemical factors. At 37 °C, the half-life of bovine prion infectivity in cell culture is 1 to 3 hours, and only 1 minute at 56 °C. However, a small number of viruses survived at 56 ° C for 60 minutes or 60 ° C for 30 minutes. After several months of storage at 4 °C , the infectivity decreased significantly. At -70 ° C for one year, the infection titer decreased. The virus is preferably stored lyophilized or stored at 2 ° C or less after addition of 2% dimethyl sulfoxide (DMSO). The inactivation of ultraviolet radiation is fast. Different strains have different pH stability, but most are inactivated at pH 4.0 or lower. The most stable pH is 7.2 to 8.0. The virus was inactivated by placing it in 20% diethyl ether chloroform at 4 ° C overnight. A prominent feature of the rinderpest virus is its very fragile glycerol, a phenomenon that has been used in the preparation of inactivated vaccines. Corruption can quickly inactivate the virus, and it may lose its infection when exposed to sunlight or naturally dry. The virus survives for a short period of time in the carcass, and the virus in the lymph nodes and spleen can survive for several weeks when stored at -25 °C. Strong base disinfection is best, glycerin, phenol, formaldehyde or β-propiolactone can quickly destroy the infectivity of rinderpest virus without significantly affecting its antigenicity. Blood coagulation The rinderpest virus has not been proven to have hemagglutination activity, but the rinderpest virus antiserum inhibits the hemagglutinin of the measles virus. The rinderpest virus also functions as a measles virus hemagglutinin receptor site that blocks the surface of the monkey red blood cells. Treating the measles virus with ether and Tween-80 can break the viral envelope into small, uniform hemagglutinin particles. This agglutination of hemagglutinin particles to monkey red blood cells can be inhibited by high titers of canine distemper or rinderpest antibodies. HeLa cells infected with measles or canine distemper virus can adsorb red blood cells of guinea pigs, but rinderpest virus cannot adsorb red blood cells. Antigenicity The antigenicity of all strains of the rinderpest virus is the same. Serological studies have shown that virions contain many soluble antigens that are not related to infectivity, including complement-binding antigens. This complement-binding antigen can be extracted from infected tissues with alcohol, acetone, or ether, and it can resist boiling for 20 minutes. There are also some precipitated antigens, two of which are thermostable; the third antigen moves the slowest during electrophoresis and is sensitive to heat. Precipitates are easily detected by using a suspension of acute diseased bovine lymph nodes and other tissues, or concentrated infected cell culture medium, and an anti-serum agar diffusion test. However, complement-binding antigens and precipitated antigens can be rapidly destroyed by spoilage. Triplex viruses are very antigenic, but each has a specific component, as evidenced by many serum tests. Cross-protection tests have shown that the immunity produced by canines infected with measles virus or rinderpest virus can resist the attack of canine distemper virus, but cattle cannot resist calves after vaccination with canine distemper virus. The hemagglutinin of the measles virus can be used as a reliable diagnostic method for detecting antibodies to calves and canine distemper. There is an infectious disease similar to rinderpest in sheep and goats in West Africa, but not transmitted to cattle. The cytopathic effect of this virus in sheep kidney cell culture is similar to that of rinderpest virus. Adapted to cell proliferation, this virus can be used to immunize cattle against rinderpest. Conversely, rinderpest antiserum can also make sheep resistant to this virus. Therefore, the pathogen of sheep disease in West Africa may be a strain of rinderpest virus, which has lost the ability to infect cattle through natural pathways, but is easily transmitted in sheep. To be continued in Part Two…
  14. The development of second-generation gene sequencing technology has pushed genomics research to a climax. Many biological problems, "measuring order" may be able to find key genes. General genome-wide sequencing covers more than 25,000 genes, compared to more than 1,400 metabolites that can be measured by total metabolomics. Although the number is small, metabolomics can be said to be getting more and more fired in the past 20 years because metabolites can more directly reflect the characteristics of biological phenotypes. Basic research method Metaboletics can be divided into two types: metabolomics and targeted metabolomics. Total metabolomics, as its name implies, detects all metabolites; while targeted metabolomics, it focuses more on certain metabolic pathways, such as the Krebs cycle, glycolysis, and so on. In this regard, Professor Fang suggested that although the coverage of total metabolomics is broader, the sensitivity is not enough; if there are some experimental or literature clues, it is better to look at targeted metabolomics. In addition, lipid metabolomics has also developed in recent years, and it belongs to the emerging field. Because of the different physical and chemical properties of lipids and most metabolites, lipid metabolomics requires different separation methods using common metabolomics. The first is to extract samples of the "medication group" and "control group", followed by gas chromatography mass spectrometry (GC-MS) or liquid phase mass spectrometry (LC-MS) to obtain data, and nuclear magnetic resonance (MRI) is relatively less used. Analysis of the data is a very important part of metabolomics. Many parameters need to be adjusted, and then statistical analysis is used to determine which metabolites have changed. Important information includes: Retention time; Accurate mass, etc. The changed data is then analyzed for metabolome pathways, typically using the KEGG database. Similarly, these databases now have little information about lipid metabolism. Academic research example Although everyone has high hopes for metabolomics to discover biomarkers, the reality is still cruel. Because the specificity of metabolites relative to disease is not strong enough, many causes may cause changes in the same metabolite, so it is difficult to determine whether a disease has been obtained through a metabolite. There are now models that judge through a set of metabolite changes, but have not yet seen clinical application. So what are the research hotspots in academia? One of the more interesting concepts is the metabolic regulation, which is about metabolite regulation of gene expression. In the past, it was thought to be the expression of gene regulatory enzymes, which in turn regulates metabolism. Some recent studies have found that the content of certain metabolites in cells affects the gene expression of cells and even cell fate. This is a process of mutual influence and is two-way. For example, last year's article in Cell found that the content of arginine changed greatly during the differentiation of immune T cells. Changing the concentration of arginine can change the fate of T cells. The other direction is to do "precise medical treatment." Different patients, while eating a drug, some people have a good effect, some people do not work well, why? Is it possible to predict the patient's drug sensitivity in advance by analyzing the metabolomics of blood samples? There are now more gene sequencing in this field, and metabolomics is still relatively rare. Of course, there is some controversy about whether metabolomics can ultimately play such a role. The last mentioned academic research direction, through the new research methods, re-write some biochemical concepts. In the past, there were many concepts, and experience was accumulated because there was no way to detect the limitations of technical conditions. And today's technology can reanalyze and study these issues. Experimental design considerations A student doing a biological study, what should you pay attention to if you want to use metabolomics to get some data? First of all, to figure out the biological problem you are studying, metabolomics can answer some questions, that is, you don't need to do this experiment. In some cases, changes in metabolomics are not particularly large, and changes may not be observed, which wastes time. Generally speaking, the major changes are metabolic diseases and tumors. In recent years, there have been many applications in the fields of immune response, stem cells, and epigenetics, but each subject researcher needs to have an in-depth understanding of the research object, and then study specific problems through metabolomics. Secondly, if it is determined that it needs to be done, communicate with the technical department before the experiment to understand each step of the sample processing to ensure that there is no error. For example, many metabolites are unstable, and the sample collection process must be low temperature; how many cells and tissues have sufficient sample volume, too small sample size will cause many metabolites to be detected; design a good control group to be reliable The comparison; there are also samples of how many repeat groups should be collected; whether you want to analyze the common metabolome or the lipid metabolome, and so on. Again, it's best to find an experienced expert to discuss how to look at metabolite changes. We recommend looking at the general changes first, and then looking at a path. If you are doing non-radioactive isotope labeling experiments, there are more to be noted: the choice of isotope labeling sites and the time of sample collection are important. Pre-experiments are generally required to determine the time required for metabolite conversion in an experimental system. This type of experiment requires relatively high equipment and is relatively expensive. It should be demonstrated in detail before the experiment. Finally, although it is a “group study” data, researchers need to have a relatively clear “hypothesis”, namely hypothesis. Because there are many metabolites of change, which metabolite changes are the most critical, it may still require experimental and literature reading to narrow the scope. Industrial application prospects There are some applications in R&D: 1 Clinically looking for biomarkers. Although there is currently no approved method for the detection of biomarkers by mass spectrometry, the market will be considerable if a reliable biomarker is developed. Because metabolomics is constantly changing compared to stable genomics, a patient may need to perform multiple tests at different stages of the disease. 2 Medically, it may replace some enzyme-linked immunosorbent assays (ELISA) because the specificity of the mass spectrometry will be higher. There are hopes to develop some routine testing items, such as expiratory diagnosis of lung cancer. 3 Develop specific nutrition programs for wealthy people. Metabolomics can detect what nutrients are missing in the body and know what dietary structure is needed to improve health. 4 Drug metabolomics. As mentioned before, it is known by metabolite changes which enzyme activities in the individual are high, thereby predicting the effects and side effects of the drug in individual patients. The industrial application of metabolomics has not seen a big market so far. Some existing companies mainly provide services to universities and research.
  15. In recent years, individualized treatments will benefit more cancer patients as the time and cost of whole genome sequencing decreases. A recent study published in Nature Medicine showed that the development of new machine learning algorithms to analyze tumor genome-wide sequence information can help predict patient prognosis and help patients choose the best treatment. Whole-genome sequencing (WGS) technology can obtain almost all genetic information of patients, including whole-genome information of cancer cells and healthy cells, which make abnormal cancer cells can be found at the level of single nucleotide, copy number, epigenetic modification, etc. To explore the clinical value of WGS, researchers at the University of Cambridge collaborated with researchers at Lund University in Sweden to develop a population-wide project called SCAN-B (Sweden Cancerome Analysis Network–Breast) (ClinicalTrials.gov ID: NCT02306096). The project has recruited breast cancer patients in Sweden since 2010 and has collected a large amount of clinical data so far. Project process In the SCAN-B project, the researchers performed a genome-wide sequencing of 254 triple-negative breast cancer (TNBC) in SCAN-B between 2010 and 2015. Triple-negative breast cancer refers to breast cancer patients with negative estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, accounting for 9% of breast cancer, and the prognosis is poor. The researchers then used a machine learning algorithm called "HRDetect" to classify the tumors. This algorithm was originally developed to detect tumors with a BRCA1/BRCA2 gene mutation signature, and mutation of either BRCA1/BRCA2 gene would greatly increase the risk of breast cancer. Currently, a new targeted drug PARP inhibitor can be used for breast cancer patients with BRCA1/BRCA2 gene mutations. According to the HRDetect algorithm, the patients were classified into three categories: high (HRDetect-high), medium (HRDetect-intermediate), and low (HRDetect-low). 59% of patients had homologous recombination repair loss (HRDetect-high): 67% of them were germline/somatic mutations of BRCA1/BRCA2, BRCA1 promoter hypermethylation, RAD51C hypermethylation or PALB2 biallelic loss. The HRDetect algorithm provides a unique diagnostic message. The researchers found that patients with high-risk (HRDetect-high) triple-negative breast cancer had the best treatment and were more sensitive to PARP inhibitors than those with low scores (HRDetect-low). Surprisingly, patients with moderate (HRDetect-intermediate) treatments had the worst treatment, and even though some patients had some better drug targets, the prognosis was still poor. Therefore, for patients with low scores, it is necessary to adopt a new treatment plan. Patients with low scores (HRDetect-low) also have poor prognosis. However, the WGS sequence information of these patients' tumors is also abnormal, which may be caused by the use of fixed drugs in clinical trials, such as immunological checkpoint inhibitors (PD-1) or AKT inhibitors. Dr. Johan Staaf, the lead author of the study, said: "Three-negative breast cancer is difficult to treat, but by genome-wide sequencing, we can identify which triple-negative breast cancer patients are more sensitive to the drugs currently used clinically. Importantly, this method allows us to provide clues for studying the mechanisms of poor prognosis and develop new drugs for such patients." With the rapid development of sequencing technology, whole genome sequencing can be completed in 24 hours, and the analysis of sequencing data can be completed in 24-48 hours. Therefore, in theory, genome-wide sequencing and analysis can be provided for each patient, and the optimal treatment plan can be selected based on the patient's own tumor genome information. “Genome-wide sequencing opens up new avenues for individualized treatment of cancer,” said co-first author Dr. Nik Zainal. “In the past, the management and analysis of large amounts of data was a major obstacle to its widespread use. But now we are reducing the time for data analysis. Let each patient get individualized treatment, which will greatly change the treatment of cancer, even some refractory cancer treatment." CD Genomics has been providing the accurate and affordable whole genome sequencing service for couple of years. The company combines both Illumina (short reads) and PacBio (long reads) platforms to achieve whole genome de novo assemblies and re-sequencing for virus, microbes, plants, animals and humans.